Bioconversion of agro-industrial wastes for the production of fibrinolytic enzyme from Bacillus halodurans IND18: Purification and biochemical characterization

Background: Agro-wastes were used for the production of fibrinolytic enzyme in solid-state fermentation. The process parameters were optimized to enhance the production of fibrinolytic enzyme from Bacillus halodurans IND18 by statistical approach. The fibrinolytic enzyme was purified, and the properties were studied. Results: A two-level full factorial design was used to screen the significant factors. The factors such as moisture, pH, and peptone were significantly affected enzyme production and these three factors were selected for further optimization using central composite design. The optimum medium for fibrinolytic enzyme production was wheat bran medium containing 1% peptone and 80% moisture with pH 8.32. Under these optimized conditions, the production of fibrinolytic enzyme was found to be 6851 U/g. The fibrinolytic enzyme was purified by 3.6-fold with 1275 U/mg specific activity. The molecular mass of fibrinolytic enzyme was determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis, and it was observed as 29 kDa. The fibrinolytic enzyme depicted an optimal pH of 9.0 and was stable at a range of pH from 8.0 to 10.0. The optimal temperature was 60°C and was stable up to 50°C. This enzyme activated plasminogen and also degraded the fibrin net of blood clot, which suggested its potential as an effective thrombolytic agent. Conclusions: Wheat bran was found to be an effective substrate for the production of fibrinolytic enzyme. The purified fibrinolytic enzyme degraded fibrin clot. The fibrinolytic enzyme could be useful to make as an effective thrombolytic agent.

Assessment of Thrombolytic Activity of Five Bangladeshi Medicinal Plants: Potential Source for Thrombolytic Compounds.

Aim: The aim of our project work was to assess the thrombolytic activity of five common Bangladeshi plant extract in different solvent. Five plants are Geodorum densiflorum (Shankhamul), Pistia stratiotes (Topa Pana), Smilax zeylanica (Kumarilata), Pandanus foetidus (Keya) & Tabernaemontana coronaria (Tagar). Plants were collected and air dried separately for three weeks. They were ground into a coarse powder. Cold extractions were performed for all plants by using different solvents. Place and Duration of Study: Department of Pharmacy, University of Chittagong and University of Science and Technology Chittagong, November, 2013. Methodology: Fresh blood was collected from healthy individuals ten volunteers (n=10). Blood was allowed to form clots in a pre-weighed sterile micro-centrifuge eppendorf tubes. After clot serum was removed and blood clot was weighed then blood clot was allowed to lysis by streptokinase. After lysis fluid was removed and the remaining of blood clot was again weighed along with the tube. Percentage of blood clot lysis was calculated on the basis of the weight difference. Weight difference of tubes obtained by weighing before and after clot lyses of blood. % clot lysis=(Weight after clot lysis/ Weight of clot before lysis)×100. This method was repeated for all extracts. Result: Among the herbs studied Pandanus foetidus (C), Pandanus foetidus (PE), Smilax zeylanica (E) and Pistia stratiotes-Root (M) showed significant % of clot lysis 47.54% 41.49%, 43.35% and 35.85% respectively with reference to standard, streptokinase (70.24%). Conclusion: These extracts lyse the blood clots In-vitro, however, we need to know In-vivo clot dissolving property. Further systemic research on these plants and may be a potential source of thrombolytic agent in future.

STUDIES ON FIBRINOLYTIC FUNCTION OF SUBTILISIN AND ITS MECHANISM / 营养学报

Objective:To evaluate the effect how Subtilisin FS33 affect thrombotic and fibrinolytic systems in vitro and in vivo. Method:Activity of Subtilisin FS33 was measured by clot liquefaction time(CLT) . On the model of 10% FeCl3 induced thrombi of carotid arteries in rats,various doses of Subtilisin FS33 were injected to the rats,and the fibrinolytic effect was observed. Results:0.5 g of the unheated blood clots gradually dissolved within 45 min,whereas the blood clots heated at 80℃ for 30 min dissolved within 3 h. This indicated that the enzyme was able to degrade blood clots in the absence of endogenous fibrinolytic factors. The experiment in vivo indicated that high dose subtilisn group could significantly prolong CT(coagulation time ) ,PT(prothrombin time) ,TT(thrombin time ) ,APTT(activated partial thromboplastin time) ,reduce ELT(euglobulin lysis time) ,decrease the content of FIB(fibrinogen) ,increase the content of FDP(fibrinogen degradation products) . D-dimer of all experimental groups waspositive. The venous thrombus in lung and kidney was dissolved totally or partly as observed by pathological section. Conclusion:Both thrombolytic effects of Subtilisin FS33 in vitro and in vivo were significant and the mechanisms might be associated with enhancing anticoagulation activity and fibrinolysis.

A Magnetic Resonance Angiographic Diagnosis and Successful Thrombolytic Therapy in The Thrombosis of Renal Vein Complicated from Nephroitc Syndrome / 대한신장학회잡지

The nephrotic syndrome has been considered a hypercoagulable state since it may be complicated by thromboembolic events of the venous or the arterial circulations. Diverse pathogenetic factors leading to the hypercoagulable state in nephrotic syndrome have been recognized. Renal vein thrombosis is a serious complication, which might lead to either renal failure or to secondary thromboembolic processes like pulmonary thromboembolism. Although it may present acutely with flank pain and macroscopic hematuria, the majority of cases run an indolent course. Until relatively recently, the diagnosis could only be confidently confirmed or excluded with selective renal venography but, more recently, computerized tomography and magnetic resonance imaging have been used. Anticoagulant therapy with heparin and warfarin apparently halts the natural progression of the disease and allowing for slow recovery. The possibility of more rapid and complete resolution with thrombolytic agents warranted their application. We described a case of bilateral renal vein thrombosis diagnosed by the new technique of magnetic resonance angiography and successful treatment by thrombolytic agent.

Early Use of Thrombolytics for Massive Pulmonary Embolism by a Comparison of Cases

Pulmonary embolism is a common condition with considerable morbidity and mortality. The diagnosis of pulmonary embolism remains a vexing problem. Prompt and accurate diagnosis is important because the mortality of untreated pulmonary embolism is high and serious complications can occur. Most physicians are inexperienced in the use of thrombolytic agents fo r pulmonary embolism, even though they utilize these agents routinely for acute myocardial infarction. We compared 2 domestic cases with 2 foreign cases. On comparison, we spent a long time in making a definite diagnosis in the domestic cases. Moreover, in the second domestic case, we did not use thrombolytic agents early, in spite of a suspected massive pulmonary embolism and then eventually he died. We conclude that bolus administration of thrombolytic agents during CPR for clinically suspected massive pulmonary embolism in emergency departments may be an acceptable technique.